It is now recognized that specific transport systems in the plasma membrane regulate the intracellular levels of essential nutrients, but very little is known about transport processes at a molecular level. We are developing well-defined systems for studying transport in mammalian cells and are using both genetic and biochemical approaches to these problems by examining transport of purines by cells growing in tissue culture. The mechanism of hypoxanthine and guanine transport by Chinese hamster lung fibroblasts is being examined by both kinetic and metabolic studies. A group of 8-azaguanine-resistant (8-AGr) sublines are used in these experiments. Some of the 8-AGr mutants are defective in hypoxanthine: quanine phosphoribosyltransferase (HGPRT), while others have normal levels of HGPRT. The former class of mutants are used for study of carrier-mediated Hyp transport in the absence of subsequent Hyp metabolism. The latter class is to be screened for mutants defective in Hyp transport. We are also developing methods for the isolation of mammalian plasma membrane vesicles that retain transport activity. Vesicles from Ehrlich ascites tumor cells are prepared by a modification of Johnstone method and utilized for study of hypoxanthine transport. This methodology will then be applied to plasma membrane vesicles isolated from parental and 8-AGr sublines of CHL cells. These latter experiments will focus on the regulatory mechanisms governing purine transport.